Chaudhary Nidhi, Agrawal Himanshu, Pandey Mamta, Onteru Suneel, Singh Dheer
Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India.
Cytotechnology. 2018 Feb;70(1):361-373. doi: 10.1007/s10616-017-0151-y. Epub 2017 Oct 14.
Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant's primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5 mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17 µg/cm collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10 days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.
小肠上皮细胞（IEC）在营养物质和毒素的吸收中起主要作用。由于牛和人类全基因组单拷贝蛋白质直系同源物具有相似性，建立反刍动物原代小肠IEC培养体系可能是毒性研究的一个有价值的工具。因此，由于印度禁止屠宰牛，当前的研究聚焦于水牛IEC培养体系的建立和特性研究。水牛空肠片段先后在生理盐水、温磷酸盐缓冲液（PBS）、含5 mM二硫苏糖醇的PBS、消化液以及含2%山梨醇的PBS中连续冲洗数次。细胞接种于涂有17 μg/cm胶原蛋白的培养板和Transwell培养板上，分别在含血清（2%胎牛血清（FBS）和10% FBS）和无血清的培养条件下培养。在50%成功的培养物中，细胞从第5天开始分化为典型的上皮鹅卵石样形态。通过上皮细胞标志物细胞角蛋白和波形蛋白以及肠细胞标志物如绒毛蛋白、闭合蛋白（ZO1）、脂肪酸结合蛋白2（FABP2）和小肠肽酶（IP）的基因表达来鉴定培养的IEC。基于形态学和基因表达谱，推荐在涂有胶原蛋白的培养板上用10% FBS培养原代水牛IEC 10天。然而，即使在涂有胶原蛋白的培养板上，50%成功的培养物在10% FBS培养条件下也未表现出上皮表型。有趣的是，与在胶原蛋白板上用10% FBS培养的分化肠细胞相比，未分化的IEC显示出FABP2、IP和ZO1转录本表达增加。因此，需要进一步研究以了解FABP2、IP和ZO1在水牛IEC分化中的作用。
